Cryptococcus neoformans is an opportunistic pathogen that is responsible for life-threatening disease in patients with AIDS or other conditions of immunocompromise. This R21 application focuses on the interactions of this facultative intracellular pathogen with the phagocytic cells of its mammalian host. These interactions resolve with varying levels of damage to the host and yeast, but the mechanisms involved are poorly understood, limiting our ability to influence resolution in favor of the host. We propose to combine RNA interference methodology and high-throughput screening assays to identify host factors that are required for the entry of C. neoformans into host cells. Aim I is to optimize methods for assessing the interactions of C. neoformans with host cells subjected to RNA interference. Aim II is to identify sequences required for fungal adherence and entry by screening RNAi libraries targeting known genes, and to confirm and prioritize these sequences for additional investigation. Results will be assessed using rigorous statistical methods and appropriate controls, and will be interpreted in the context of our expertise in the biology of Cryptococcus and its host. The long-term goal of our studies is to understand the pathogenesis of cryptococcosis. The proposed studies will elucidate a crucial aspect of pathogenesis that determines survival and dissemination of the organism and the host's ability to limit disease. This work will additionally open new areas for investigation of an important pathogen and develop experimental tools that can be readily applied to other areas of pathogen biology. PUBLIC HEALTH RELEVANCE: This research is highly relevant to public health because the organism under study causes serious human illness for which current therapies are not adequate. The interactions of Cryptococcus neoformans with host cells are fundamental to its ability to cause disease, so understanding and inhibiting these processes may advance treatment of cryptococcal infection. Further, this work will contribute to basic science knowledge, which will provide insights into pathogenic microbes as well as other areas of biology.